1. Field of the Invention
The invention relates to a method of predicting disease-free survival probability in cancer patients. In particular, the method enables prediction of tumor recurrence in node-negative breast cancers as related to the levels and the number of stress response proteins in primary tumors.
2. Description of Related Art
Stress response proteins, srp's, have been recognized for several years, although in earlier terminology they were commonly called heat shock proteins, hsp's, because they were originally discovered as families of related proteins rapidly overproduced in divergent species in response to temperature stress. Subsequently these proteins were found to be induced in response to a variety of environmental stresses, including stimuli such as heat, heavy metals, toxins, drugs, hypoxia, and alcohol.
The precise function of stress response proteins is still largely a matter of speculation. It is widely assumed that these proteins protect cells from the effects of stress, but little is known about the mechanisms of induction and even less is understood about the relationships between number and amount of protein induced and the underlying physiological phenomenon.
There have been speculations that pretumorous or tumor cells might express increased amounts of stress response proteins, leading some workers to search for a relation between levels of these proteins and tumor manifestation. But although readily induced, the higher levels of heat stress proteins did not appear to relate to increased probability of tumor recurrence; in fact, some studies indicated that metastatic tumor burden generally decreased following induction of stress proteins (S. P.. Tomasovic and D. R. Welch, Hyperthermia 2, 253 (1986)). Subsequent work by McGuire and colleagues, however, demonstrated that an estrogen-induced protein found in MCF-7 human breast cancer cells was identical to one of the earlier discovered heat shock proteins, hsp27, and that hsp27 might be associated with node-negative breast cancer patients at high risk of recurrence. Nevertheless, correlation factors were relatively weak and not sufficient to suggest a clinically useful method of prognostication.
The phenomenon of heat shock response was first observed nearly three decades ago by Ritossa (F. Ritossa, Experientia 18, 571 (1962)) who found that an increase in temperature from 20.degree. to 37.degree. C., as well as exposure to certain chemicals such as dinitrophenol or sodium salicylate, leads to a remarkable change in the puffing pattern of polytene chromosomes in salivary glands of fruit flies (Drosophila hydei). Nearly 12 years after this observation, Tissieres et al. (A. Tissieres, H. K. Mitchell, U. M. Tracy, J. Mol. Biol. 84, 389 (1974)) reported the induction of a set of proteins called heat shock proteins (hsp's), as a consequence of heat shock. Today, practically all types of organisms are known to respond to an increase in temperature in a basically similar fashion by massive synthesis and accumulation of a group of hsp's with almost no tissue or cell type specificity. Major hsp's are now known to be very highly conserved through evolution, strongly suggesting their vital role in survival of the organisms. Nearly all species induce the synthesis of proteins in the size ranges of 80 to 90 kDa, 68 to 74 kDa, and 18 to 30 kDa. In the past few years, the genes encoding the hsp's have been isolated and through sequence analysis have been placed into three "universal" families. These are known by their molecular weights: hsp90, hsp70, and hsp27 (the exact molecular weight differs slightly from organism to organism). These hsp genes contain a conserved sequence of 14 base pairs in the 5' noncoding region, the Pelham box, which serves as the promoter for hsp mRNA transcription. Recently, a relationship between the sequences of hsp' s and another family of stress proteins, the glucose-regulated proteins (grp's), has been reported. These proteins are oversynthesized in response to glucose starvation. Two major grp's have been identified as grp94 and grp78.
Although the precise function of stress response proteins (srp's) is not known, they are thought to be intimately involved in enhancing the cell's ability to recover from stress (e.g. conferring thermotolerance). Yet the exact biochemical mechanism of this protection of cells against physical and chemical environmental insults remains a mystery. There are several excellent and comprehensive reviews on this subject including the organization and regulation of expression of hsp genes (S. Linquist, E. A. Craig, Ann. Rev. Genet. 22, 631 (1988) M. J. Schlesinger, J. Cell Biol. 103, 321 (1986); H.R.B. pelham, Cell 46, 959 (1988); E. A. Craig, CRC Crit. Rev. Biochem. 18, 239 (1985)).
In 1980, a cytoplasmic, estrogen-induced protein of 24,000-28,000 Daltons molecular weight (termed "24K") in MCF-7 human breast cancer cells (D. P. Edwards, D. J. Adams, N. Savage, W. L. McGuire, Biochem. Res. Commun. 93, 804 (1980)) was reported. Its relative abundance in estrogen-stimulated MCF-7 cells enabled researchers to rapidly develop a highly specific monoclonal antibody against it (D. J. Adams, H. Hajj, D. P. Edwards, R. J. Bjercke, W. L. McGuire, Cancer Res. 43, 4297 (1983)). Nucleotide and deduced amino acid sequence of "24K" (S. A. W. Fuqua, M. B. Salingaros, W. L. McGuire, ibid. 49, 4126 (1989)) revealed its identity to the low molecular weight human heat shock protein hsp27, earlier reported in HeLa cells (E. Hickey et al., Nucleic Acid Res. 14, 4127 (1986)). Somatic cell hybridization showed that it is a multigene family, located on three different chromosomes namely 3,9 and X (S. McGuire, S. A. W. Fuqua, S. L. Naylor, D. A. Helen-Davis, W. L. McGuire, Somatic Cell Genet. 15, 167 (1989)). It is dually induced by heat shock as well as by estrogen in MCF-7 cells. A study of its possible significance for predicting clinical outcome showed that it was a factor for defining node-negative breast cancer patients at high risk of recurrence (G. C. Chamness, A. Ruiz, L. Fulcher, G. M. Clark, W. L. McGuire, Breast Cancer Res. Treat. 12, 130 (1988) (Abstract #94); G. C. Chamness et al., Proc. Am. Assoc. Cancer Res. 30, 252 (1989)) (Abstract #1002).
The heat shock protein hsp90 is known to interact with several protein-tyrosine kinases between the time of their synthesis and their ultimate association with the plasma membrane. The transforming protein of Rous Sarcoma Virus, pp60.sup.src, was the first tyrosine kinase with which hsp90 was shown to have a specific association (J. S. Brugge, E. Erikson, R. L. Erikson, Cell 25, 363 (1981); H. Oppermann, W. Levinson, J. M. Bishop, Proc. Natl. Acad. Sci. 78, 1067 (1981)). Other transforming proteins with tyrosine kinase activity, yes, fps, fes, and fgr, also form stable complexes with a 90 kDa protein. In some cases this 90 kDa protein has been identified as hsp90 (B. Adkins, T. Hunter, B. M. Sefton, J. Virol. 43, 448 (1982); L. A. Lipsich, J. R. Cutt, J. S. Brugge, Mol. Cell Biol. 2, 875 (1982); and A. Ziemiecki, Virology 151, 265 (1986)). It has been proposed that hsp90 transports and modulates these kinases by forming soluble, inactive complexes. Hsp90 has also been found associated with other cellular kinases, e.g. heme-controlled elF2-alpha kinase and casein kinase II (D. W. Rose, R. E. H. Wettenhall, W. Kudlicki, G. Kramer, B. Hardesty, Biochemistry 26, 6583 (1987)).
All steroid hormone receptors, including the estrogen, progesterone, and glucocorticoid receptors, can be isolated in the inactivated state (i.e.. in the absence of steroid hormones) as approximately 300 kDa complexes, which in addition to the specific hormone-binding proteins, contain 90 kDa proteins that have now been identified as hsp90 (M. G. Catelli, C. Radanyi, J. M. Renoir, N. Binart, E. E. Baulieu, J. Cell Biochem. Suppl. 12D. 286 (1988); J. J. Dougherty, R. K. Puri, D. O. Toft, J. Biol. Chem. 259, 8004 (1984); I. Joab, et al., Nature 308, 850 (1984); G. Redeuilh, B. Moncharmont, C. Secco, E.-E. Baulieu, J. Biol. Chem. 262, 6969 (1987); J.-M. Renoir, T. Buchou, E.-E. Baulieu, Biochemistry 25, 6405 (1986); and E. R. Sanchez, P. R. Housley, W. B. Pratt, J. Steroid Biochem. 24, 9 (1986)). Dissociation of hsp90 from the complex leads to the activation of the receptor for DNA binding (I. Joab, et al., Nature 308, 850 ( 1984); J.-M. Renoir, T. Buchou, E.-E. Baulieu, Biochemistry 25, 6405 (1986); and E. R. Sanchez, et al., J. Biol. Chem. 262, 6986 (1987)). In the absence of hsp90, the hormone-binding receptor will bind to the DNA whether hormone is present or not (E. R. Sanchez, et al., J. Biol. Chem. 262, 6986 (1987)). Hsp90 itself binds neither DNA nor hormone. Apparently, binding of hsp90 to the receptor prevents the receptor from binding to DNA until hormone disrupts association of hsp90 to the receptor.
In broad outline, hsp90 appears to play a role in steroid receptor complexes similar to that in tyrosine kinase complexes, keeping the receptor inactive until the proper signal for activation is received.
Recently, hsp90 has also been reported to associate with actin in lymphocyte extracts, in a manner dependent on calcium and regulated by calmodulin (S. Koyasu, et al., Proc. Natl. Acad. Sci. USA 83, 8054 (1986); and E. Nishida, S. Koyasu, H. Sakai, I. Yahara, J. Biol. Chem. 261, 16033 (1986)). It has been postulated that the actin association provides a mechanism for transport of hsp90. In this regard, and considering the tendency of hsp90 to move into the nucleus with heat shock, it is notable that actin filaments rearrange during heat shock and may even be found in substantial quantities in the nuclei of heat-shocked cells (W. J. Welch, J. P. Suhan, J. Cell Biol. 101, 1198 (1985)). Hsp90 also appears to be associated with tubulin both in vitro and in vivo (E. H. Bresnick, T. Redmond, E. R. Sanchez, W. B. Pratt, M. J. Welsh, J. Cell Biochem. Suppl. 12D, 283 (1988)). Given the high concentrations of actin, tubulin, and hsp90 in the cell, these associations may be biologically significant.
A tumor-specific transplantation antigen, Meth A, has also recently been identified as hsp90 (S. J. Ullrich, E. A. Robinson, L. W. Law, M. Willingham, E. Apella, Proc. Natl. Acad. Sci. USA 83, 121 (1986)).
In humans, the heat shock protein hsp70 represents a multigene family, located on chromosomes 6, 14, 21, and at least one other chromosome ( A. M. Goate, et al., Hum. Genet. 75, 123 (1987); and G. S. Harrison, et al., Somatic Cell Mol. Genet. 13, 119 (1987)). Their protein products are present in different cellular compartments and are often associated with other proteins. All bind ATP with high affinity (T. G. Chappell, et al., Cell 45, 3 (1986); W. J. Welch, J. R. Feramisco, Mol. Cell. Biol. 5, 1229 (1985); and M. Zylicz, J. H. LeBowitz, R. McMacken, C. P. Georgopoulos, Proc. Natl. Acad. Sci. USA 80, 6431 (1983)) and are implicated in a number of cellular processes. The major hsp70 is a cell cycle regulated protein (K. L. Milarski, R. Morimoto, Proc. Natl. Acad. Sci. USA 83, 9517 (1986)), is serum stimulated (B. J. Wu, R. I. Morimoto, Proc. Natl. Acad. Sci. USA 82, 6070 (1985)), and is induced by adenovirus EIA protein (J. R. Nevins, Cell 29, 913 (1982)).
It seems that the cell exploits a general property of the hsp70 family, namely the ability to disrupt protein-protein interactions, to perform specific tasks. Most of the "reactions" involving proteins of the hsp70 family require ATP. Probably the disruption of the protein-protein interactions uses the energy released on ATP hydrolysis.
One function of hsp70 may be the repair of damaged cells. Very shortly following heat shock, hsp70 translocates from cytoplasm to nucleus changing from a "soluble" cytosolic to an "insoluble" nuclear-matrix form. In the nucleus it subsequently concentrates in nucleoli where it apparently binds to partially assembled ribosomes (W. J. Welch, J. P. Suhan, J. Cell Biol. 103, 2035 (1986)). Nucleoli are very sensitive to thermal damage, but transfection of cells with a plasmid that overproduces hsp70 accelerates their recovery from heat shock (H.R.B. Pelham, EMBO J 3, 3095 (1984)), indicating that hsp70 binds to denatured or abnormal proteins after heat shock to prevent their aggregation and thus to prevent cellular damage. Hsp70 is rapidly and completely released from "insoluble" nuclear matrix on addition of ATP (M. J. Lewis, H. R. B. Pelham, EMBO J 4, 3137 (1985)). This observation led Lewis and Pelham (M. J. Lewis, H.R.B. Pelham, EMBO J 4, 3137 (1985)) to propose that hsp70 binds to denatured, aggregated proteins and solubilizes them. Energy from ATP hydrolysis subsequently causes hsp70 to release, thereby allowing the proteins to refold.
Clathrin-uncoating ATPase has been identified as a member of the hsp70 gene family. In the presence of ATP, an hsp70-like protein binds to the clathrin cages and is induced to hydrolyze ATP, resulting in the disruption of clathrin-clathrin interactions and finally in disassembly of the cage into clathrin trimers (J. E. Rothman, S. L. Schmid, Cell 46, 5 (1986); T. G. Chappell, et al., Cell 45, 3 (1986); and E. Ungewickell, EMBO J 4, 3385 (1985)).
In E. coli, hsp70 is the product of the dnak gene, which encodes a protein that is 50% identical in amino acid sequence to hsp70 of eukaryotes (J. C. A. Bardwell, E. A. Craig, Proc. Natl. Acad. Sci. USA 81, 848 (1984)). Dnak protein interacts with lambda phage 0 and P proteins during phage replication (J. H. LeBowitz, C. Zylicz, C. Georgopoulos, R. McMacken, Proc. Natl. Acad. Sci. USA 82, 3988 (1985); and Dodson et al., Proc. Natl. Acad. Sci. USA 82, 4678 (1985)), again implicating hsp70 in the disruption of a tight protein-protein interaction. Like clathrin uncoating, this is an example of a specific function that exploits the general properties of the hsp70-like proteins.
In cells that overproduce the transformation-associated protein p53, stable complexes form between p53 and hsp70-related proteins (0. Pinhasi-Kimhi, D. Michalovitz, A. Ben-Zeev, M. Oren, Nature 320, 182 (1986)). Mutations in the gene encoding p53 that inactivate its tumor suppressing potential also result in the synthesis of mutant proteins which show preferential association with hsp70-like proteins and have an increased half-life (C. A. Findley, et al., Mol. Cell. Biol. 8, 531 (1988)). It is hypothesized that this interaction leads to a higher stability of p53, and the complex can be dissociated in vitro with ATP. Interestingly, p53 synthesized in E. coli is found in association with dnak protein (C. F. Clarke, et al., Mol. Cell. Biol. 8, 1206 (1988)).
Several eukaryotic cell DNA viruses, i.e., adenovirus (J. R. Nevins, Cell 29, 913 (1982)), herpes Virus (E. L. Notarianni, C. M. Preston, Virology 123, 113 (1982)), and Simian Virus 40 and polyoma viruses (E. W. Khanjian, H. Turler, Mol. Cell. Biol. 3, 1 (1983)) activate synthesis of hsp70 early in infection. Newcastle Disease Virus, an RNA virus, induces hsp70 and hsp90 in infected chicken cells (P. C. Collins, L. E. Hightower, J. Virol. 44, 703 (1982)). Hsp70 itself is reported to have a protease activity (H. K. Mitchell, N. S. Petersen, C. H. Buzin, Proc. Natl. Acad. Sci. USA 82 4969 (1985)).
Two proteins related to hsp70 and hsp90 and regulated by glucose starvation have been identified as grp78 and grp94, respectively (A. S. Lee, J. Bell, J. Ting, J. Biol. Chem. 259, 4616 (1984); and R. P. C. Shiu, J. Pouyssegur, I. Pastan, Proc. Natl. Acad. Sci. USA 74, 3840 (1977)). Grp's are not normally heat-inducible, but are overproduced under a variety of other physiological stresses such as anoxia, paramyxovirus infection, and treatment of cells with glycosylation inhibitors (S. C. Chang, et al., Proc. Natl. Acad. Sci. USA 84, 680 (1987)) or the calcium ionophore A23187 (Lin et al., Mol. Cell Biol. 6, 1235 (1986)). These proteins are abundant in secretory cells and are found associated with endoplasmic reticulum, and may possibly carry out the same functions as hsp's.
Grp78 is about 60% homologous to hsp70 and is identical to BiP (S. Munro, H. Pelham, Cell 46, 291 (1986)), a protein known to bind to the immunoglobulin heavy chains in pre-B cells that do not make light chains (Bole et al., J. Cell Biol. 102, 1558 (1986)). This finding suggests that grp78 prevents the formation of heavy chain aggregate and thus helps the process of immunoglobulin assembly. Grp78 binds to the aberrant proteins to keep them soluble in the same way as hsp70 acts on heat-denatured nuclear proteins. For example, it associates with mutants of hemagglutinin of influenza virus that fail to assemble into a mature trimeric glycoprotein (M. J. Gething, K. McCammon, J. Sambrook, Cell 46, 939 (1986)). Mammalian cell lines with decreased amounts of grp78 show increased secretion of mutant proteins (A. Dorner, M. Krane, R. Kaufman, J. Cell. Biochem. Suppl. 12D, 276 (1988)).
Grp94 has been partially sequenced, showing that the protein is more than 50% homologous to yeast hsp90 and Drosophila hsp83 (P. K. Sorger, H.R.B. Pelham, J. Mol. Biol. 194, 341 (1987)). It is glycosylated, soluble in the absence of detergents, and is probably a luminal protein. The role of grp94 is even less understood than that of grp78.
There is also little information on the function of the low molecular weight hsp's. A stretch of 75 amino acids which are conserved among four small Drosophila hsp's is found to be 50% homologous to the B chain of mammalian lens alpha crystallins (H. Bloemendal, T. Berns, A. Zweers, H. Hoenders, E. L. Benedetti, Eur. J. Biochem. 24, 401 (1972); and T. D. Ingolia, E. A. Craig, Proc. Natl. Acad. Sci. USA 79, 2360 (1982)), suggesting that these hsp's may serve some kind of structural role. The Drosophila hsp's form large insoluble aggregates in a perinuclear region of the cell after prolonged heat shock (N. C. Collier, M. J. Schlesinger, J. Cell Biol., 103, 1495 (1986); and L. Nover, K.-D. Scharf, D. Neumann, Mol. Cell. Biol. 3, 1648 (1983)), but these aggregates dissociate during cell recovery.
There is also an association of srp's with acquired drug resistance. Exposure of renal adenocarcinoma cells to heat shock or chemical stresses has shown that the major MDR1 gene promoter has heat shock elements and its expression (both mRNA and protein) is increased by these stresses, with a concomitant development of resistance to vinblastine (K.-V. Chin, S. Tanaka, G. Darlington, I. Pastan, M. M. Gottesman, J. Biol. Chem. 265, 221 (1990)). MDR1 RNA levels, however, did not change following stresses that normally induce grp's. Similarly, in Chinese hamster ovary cells, Shen et al. (J. Shen, et al., Proc. Natl. Acad. Sci. USA 84, 3278 (1987)) found that the induction of grp's did not change the level of MDR1-encoded P-glycoprotein. But these cells nevertheless acquired resistance to doxorubicin through an unknown mechanism. These observations suggest that some srp's may also be involved either indirectly or directly in conferring drug resistance to cells. In renal cells, MDR1-encoded P-glycoprotein may additionally protect cells from the effects of heat shock and chemical stresses.
More recently, Huot et al. (personal communication) found that transfection of Chinese hamster ovary cells with the hsp27 gene results in development of multidrug resistance. These studies indicate the role of a specific hsp in the phenomenon of multidrug resistance.
Prognosis in clinical cancer is an area of great concern and interest. It is important to know the aggressiveness of the malignant cells and the likelihood of tumor recurrence in order to plan the most effective therapy. Breast cancer, for example, is managed by several alternative strategies. In some cases local-regional and systemic radiation therapy is utilized while in other cases mastectomy and chemotherapy or mastectomy and radiation therapy are employed. Current treatment decisions for individual breast cancer patients are frequently based on (1) the number of axillary lymph nodes involved with disease, (2) estrogen receptor and progesterone receptor status, (3) the size of the primary tumor, and (4) stage of disease at diagnosis (G. M. Clark et al., N. Engl. J. Med. 309. 1343 (1983)). It has also been reported that DNA aneuploidy and proliferative rate (percent S-phase) can help in predicting the course of disease (L. G. Dressler et al., Cancer 61, 420 (1988); and G. M. Clark et al., N. Engl. J. Med. 320, 627 (1989)). However, even with these additional factors, we are still unable to accurately predict the course of disease for all breast cancer patients. There is clearly a need to identify new markers, in order to separate patients with good prognosis who will need no further therapy from those more likely to recur who might benefit from more intensive treatments.
This is particularly true in the case of breast cancer which has not progressed to the axillary lymph nodes. There is now evidence in prospective randomized clinical trials that adjuvant endocrine therapy and adjuvant chemotherapy beginning immediately after surgical removal of the primary breast tumor can be of benefit in some of these node-negative patients. This has led to official and unofficial recommendations that most if not all node-negative breast cancer patients should be considered for some form of adjuvant therapy. But since the majority (.about.70%) of these patients enjoy long-term survival following surgery and/or radiotherapy without further treatment, it may be inappropriate to recommend adjuvant therapy for all of these patients. If there were sufficiently good methods to distinguish those node-negative patients who are "cured" from those destined to recur, only the latter should be treated. Thus, there is a great need for a general method of predicting tumor recurrence in these patients and in cancer patients in general once the primary tumor is detected.
The present invention is a significant step in the ability to predict with some confidence the likelihood of cancer recurrence. It is clear from the extensive studies on the stress response proteins that they have an important physiological role, but until now no one has been able to relate their levels or presence to clinical manifestations of dysfunction. Now the survival risk to cancer patients can be better assessed and aggressive therapies applied as indicated to those in high risk groups.
The present invention is the discovery that the number and level of stress response proteins in primary tumor tissue show an unexpected and surprising correlation with tumor recurrence. Consequently, the present work represents a significant advancement in cancer management because early identification of patients at risk for tumor recurrence will permit aggressive early treatment with significantly enhanced potential for survival.